The present invention relates to active fragments of collapsin response mediator protein-1 (CRMP-1), methods for treating cancer with active fragments of hCRMP-1, and methods for inhibiting the proliferation of cancer using CRMP-1 and active fragments of CRMP-1.
Collapsin response mediator protein-1 (CRMP-1), also named as dihydropyrimidinase related protein-1 (DRP-1), is a 62 kDa phosphoprotein. CRMP-1 was originally discovered in the brain tissue and thought to be a brain specific protein involved in the collapsin-induced growth cone collapse during neural development. Torres et al., DNA Res., 5(6): 393-395 (1998).
Collapsin response mediator proteins (CRMPs) belong to a family of phosphoproteins, which mediate semaphorin/collapsin-induced growth cone collapse and are believed to be involved in both axonal guidance and neuronal differentiation. CRMPs are expressed mainly in the nervous system, especially during embryogenesis. Immunocytochemical studies have shown that CRMPs are distributed in the lamellipodia and filopodia of the growth cone, the shaft of axons, and the neuronal cell body. Their expression and phosphorylation are spatially and temporally regulated during development although their molecular mechanisms of action are yet to be clearly.
The members of CRMPs bear the sequence homology to UNC-33, a nematode protein, whose absence produces aberrant elongation of axons and uncoordinated movement in the worm Caenorhabditis elegans. Li et al., Genetics, 132(3): 675-689 (1992). CRMP family members have a 50%-70% amino acid sequence homology. Five members of the CRMP gene family (crmp-1, crmp-2, crmp-3, crmp-4, and crmp-5), encoding closely related 60-66 kDa proteins, have been independently cloned by various laboratories. Each CRMP is believed to have a unique function. The members of the CRMP family have been referred to as CRMP (collapsin response mediator protein), TOAD-64 (turned on after division of a 64 kD protein), Ulip (UNC-33 like phosphoprotein), DRP (dihydropyrimidinase related protein) and TUC (TOAD/Ulip/CRMP). Nonetheless, the most frequently used name in medical literature is CRMP.
Transcription of the ORMP gene is differentially regulated. Inaaaki et al., Histochem. Cell Biol., 113: 37-41 (2000); Matsuo et al., J. Biol. Chem., 275(22): 16560-16568 (2000); Quach et al., Gene, 242(1-2): 175-182 (2000). Mouse CRMP-1, CRMP-4 and CRMP-5 are mainly expressed in the fetal brain and not in the brain of the adult mice. On the other hand, CRMP-2 and CRMP-3 are expressed in the brain of both the fetal and the adult mice. However, in the adult mice, CRMP-3 is localized in the cerebellum. In PC-12 cells, after induction of neuronal differentiation by nerve growth factor (NGF), CRMP-4 was strongly up-regulated, whereas CRMP-1 and CRMP-2 only increased slightly and CRMP-3 was down-regulated. Byk et al., Eur. J. Biochem. 254:14-24(1998). At this time, only the promoter of human CRMP-4 has been isolated and analyzed. Matsuo et al., J. Biol. Chem., 275(22): 16560-16568 (2000). No studies of the regulatory elements of other members of the CRMP family have been conducted.
Recent works reported that the level of expression of the gene encoding human CRMP-1 (hereinafter “hCRMP-1 gene”) inversely affects cancer invasion and metastasis, (i.e., the higher the level of expression, the lower the incidence of cancer invasion and metastasis) and thus characterized the human CRMP-1 gene as an invasion-suppression gene. The following studies found that low-expression patients of hCRMP-1 had more advanced diseases and lymph node metastases, while high-expression patients of hCRMP-1 had a significantly longer disease-free and overall survival period. Shih et al., J. Natl. Cancer Inst., 93(18): 1392-1400 (2001); Chu et al., Am. J. Respir. Cell Mol. Biol., 17: 353-360 (1997); and Shih et al., Clinical & Exper. Metastasis, 20: 69-76 (2003).
However, the reports cited above only examined the biological activities of full-length hCRMP-1 protein; the effect of fragments of hCRMP-1 was unknown and the active portions of the full-length protein had not been identified. Further, the prior art only reported the effect of CRMP-1 on metastasis and invasion; any effect on cell proliferation had not been identified.
In accordance with the present invention, certain active fragments have now been discovered and are advantageous in that they can be produced much more easily than the full-length CRMP-1 and in larger quantities, yet still retain all or most of the original biological activities against tumor cells. This would make commercial production more economically and technically feasible.
In addition, it is surprising that certain active fragments achieve better and broader antiproliferative effect on cancer cells than the full-length CRMP-1. For example, CN3, as discussed herein, shows certain improved results over full-length hCRMP-1. Other fragments also exhibit better selectivity in antiproliferative effect on cancer cells than the full-length CRMP-1. For example, the CN5 and CN7 fragments showed good selectivity in inhibiting growth of lung cancer cells while not affecting the growth of normal cells. Such selectivity was not observed when using the full-length CRMP-1. In addition, where the full-length CRMP-1 protein showed suppression of growth of normal cells, certain fragments such as CN5 showed no such side effect, further illustrating that these fragments have advantages over the prior art. There has thus been a long-felt and unfulfilled need in the art to identify active fragments of CRMP-1 so as to allow the large-scale production of protein fragments having the same or better activity as the full-length protein.
Additionally, there has been a need to identify additional agents that act on cell proliferation. As discussed above, it was previously believed that full-length CRMP-1 did not inhibit cell proliferation based on studies in lung cancer cells; however, it has now been determined that full-length CRMP-1 inhibits proliferation in prostate cancer, colon cancer, and breast cancer. It has also been determined that, despite prior studies, full-length CRMP-1 inhibits lung cancer cells to a lesser extent. For example, using poorly differenciated human lung adenocarcinoma cells established from a patient, Shih et al. observed no antiproliferative activity of full-length CRMP-1, see J. Natl. Cancer Inst., 93(18): 1392-1400 (2001). In contrast, our experiments showed full-length CRMP-1 has antiproliferative activity towards the human lung large-cell carcinoma cells H460.